PROJECT SUMMARY: A primary function of growth plate cartilage is to support bone formation and elongation during endochondral ossification. Growth plate chondrocytes undergo rapid proliferation and matrix synthesis followed by hypertrophic differentiation. Hypertrophic chondrocytes secrete various factors that degrade the cartilage matrix, recruit vascular cells and osteoblast progenitors, and promote the differentiation of osteoblasts responsible for new bone formation. Dogma dictates that hypertrophic chondrocytes ultimately undergo apoptosis to facilitate removal of the cartilage template that is eventual replaced by bone. Recent cartilage-specific lineage tracing studies have suggested that terminal hypertrophic chondrocytes are capable of directly differentiating into mature osteoblasts during bone formation and repair via a process known as transdifferentiation. However, our preliminary data suggests that at least a subset of terminal hypertrophic chondrocytes undergo dedifferentiation to generate bone marrow mesenchymal stem/progenitor cells (BMSCs) capable of differentiating into various mature cell types including: osteoblasts and adipocytes. Since almost nothing is known about this process, our long-term goal is to identify the cellular and molecular mechanism(s) that regulate hypertrophic chondrocyte dedifferentiation during endochondral bone formation. Using a variety of sophisticated mouse genetic models and in vitro systems, we aim to: (Aim 1) identify whether hypertrophic chondrocytes dedifferentiate to form a molecularly and functionally distinct population of multipotent BMSCs, (Aim 2) determine whether NOTCH signaling in hypertrophic chondrocytes is necessary and/or sufficient to promote chondrocyte dedifferentiation during endochondral bone formation, and (Aim 3) establish whether SOX2 is an important regulator of hypertrophic chondrocyte dedifferentiation and a target of NOTCH signaling in the regulation of this process. Completion of these aims will have broad implications in skeletal biology by elucidating fundamental cellular and molecular mechanisms associated with the novel process of hypertrophic chondrocyte dedifferentiation during endochondral ossification. This work will also aid our understanding of NOTCH-related skeletal diseases, as well as, set the stage for developing novel approaches for the ex vivo generation of mesenchymal stem/progenitors from cartilage for use in regenerative medicine or cell therapeutic applications.